Accessory heterozygous mutations in cone photoreceptor CNGA3 exacerbate CNG channel-associated retinopathy

Abstract

Mutations in CNGA3 and CNGB3, the genes encoding the subunits of the tetrameric cone photoreceptor cyclic nucleotide-gated ion channel, cause achromatopsia, a congenital retinal disorder characterized by loss of cone function. However, a small number of patients carrying the CNGB3/c.1208G>A;p.R403Q mutation present with a variable retinal phenotype ranging from complete and incomplete achromatopsia to moderate cone dysfunction or progressive cone dystrophy. By exploring a large patient cohort and published cases, we identified 16 unrelated individuals who were homozygous or (compound-)heterozygous for the CNGB3/c.1208G>A;p.R403Q mutation. In-depth genetic and clinical analysis revealed a co-occurrence of a mutant CNGA3 allele in a high proportion of these patients (10 of 16), likely contributing to the disease phenotype. To verify these findings, we generated a Cngb3R403Q/R403Q mouse model, which was crossbred with Cnga3-deficient (Cnga3-/-) mice to obtain triallelic Cnga3+/- Cngb3R403Q/R403Q mutants. As in human subjects, there was a striking genotype-phenotype correlation, since the presence of 1 Cnga3-null allele exacerbated the cone dystrophy phenotype in Cngb3R403Q/R403Q mice. These findings strongly suggest a digenic and triallelic inheritance pattern in a subset of patients with achromatopsia/severe cone dystrophy linked to the CNGB3/p.R403Q mutation, with important implications for diagnosis, prognosis, and genetic counseling.

Keywords: Genetics; Molecular genetics; Ophthalmology; Retinopathy.

Figure 1. Pedigrees of patients and segregation of CNGB3 and CNGA3 disease alleles.

Pedigrees are subdivided into 3 groups according to genotypes and correlated disease severity. Group 1: Compound heterozygosity for CNGB3/p.R403Q and a protein truncation mutation in CNGB3 and no mutation in CNGA3. Group 2: Homozygosity for the CNGB3/c.1208G>A;p.R403Q mutation and an additional heterozygous CNGA3 mutation. Group 3: Compound heterozygosity for CNGB3/c.1208G>A;p.R403Q and a protein truncation or splicing mutation in CNGB3 and an additional heterozygous CNGA3 mutation. The genotypes for CNGB3 and CNGA3 of all tested individuals are provided. The presence of 2 mutant CNGB3 alleles with homozygosity for the CNGB3/c.1208G>A;p.R403Q mutation was confirmed by qRT-PCR. Notably, siblings and parents carrying single heterozygous mutations in both CNGB3 and CNGA3 (CHRO208-I:2; CHRO251-I:1; CHRO979-III:1, and CHRO1050-I:1) are unaffected. The different mutations (M1-M3) in each family are defined above the respective pedigree. M1 was always selected for the p.R403Q mutation.

Figure 2. Cone function loss in aged Cngb3^R403Q/R403Q and Cnga3^+/– Cngb3^R403Q/R403Q mutants.

(A) Results of the ERG recordings of WT (black) and Cngb3^R403Q/R403Q mice (red) at 1 month (M1) and 7–12 months (M7–12) of age (n = 3–5). Furthermore, mice were tested on 2 genetic backgrounds (129/Sv × C57BL/6N [n = 4] and 129/Sv [n = 3]). (B) Results of the ERG recordings of Cnga3^+/– (black) and Cnga3^+/– Cngb3^R403Q/R403Q mice (red) (n = 3). ERG data are presented as box-and-whisker plots (boxes: 25%–75% quantile range, whiskers: 5% and 95% quantiles, asterisks: median).

Figure 3. Cone degeneration and reduced CNGA3 and CNGB3 immuno staining in aged Cngb3^R403Q/R403Q and Cnga3^+/– Cngb3^R403Q/R403Q mutants.

Immunofluorescence of cryosections from murine retinas (n = 3 per genotype) at 7–12 months of age (WT, Cnga3^+/– Cngb3^+/+, Cngb3^R403Q/R403Q, and Cnga3^+/– Cngb3^R403Q/R403Q). Scale bars: 20 μm. S-opsin and cGMP staining with the respective antibodies was performed simultaneously on the same slices and imaged by multicolor laser scanning confocal micrography. Antibodies were diluted as follows: cGMP: 1:3,000; cone arrestin: 1:500; S-opsin: 1:300; M-opsin: 1:300; and CNGB3-antiserum: 1:5,000. INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment.